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Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Snow et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC., Competing Interests: There are no competing interests to declare., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based ‘gain of function’ assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions, including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. In addition, different strategies are currently being tested to scale up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcγRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo . However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD4 + T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 MAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted.

To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells. IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC., (Copyright © 2019 American Society for Microbiology.)

The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a "closed" conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands. IMPORTANCE Emerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials., (Copyright © 2018 Richard et al.)

It has been known for more than 30 years that HIV-1 infection drives a very potent B cell response resulting in the production of anti-HIV-1 antibodies targeting several viral proteins, particularly its envelope glycoproteins (Env). Env epitopes are exposed on the surfaces of viral particles and infected cells where they are targets of potentially protective antibodies. These antibodies can interdict infection by neutralization and there is strong evidence suggesting that Fc-mediated effector function can also contribute to protection. Current evidence suggests that Fc-mediated effector function plays a role in protection against infection by broadly neutralizing antibodies and it might be important for protection by non-neutralizing antibodies. Fc-mediated effector function includes diverse mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation, antibody-dependent cellular phagocytosis, antibody-dependent cell-mediated virus inhibition, antibody-mediated trancytosis inhibition, and antibody-mediated virus opsonization. All these functions could be beneficial in fighting viral infections, including HIV-1. In this perspective, we discuss the latest developments in ADCC research discussed at the HIVR4P satellite session on non-neutralizing antibodies, with emphasis on the mechanisms of ADCC resistance used by HIV-1, the structural basis of epitopes recognized by antibodies that mediate ADCC, natural killer-cell education and ADCC, and murine models to study ADCC against HIV-1.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals. IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection., Importance: This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

It is widely held that HIV-1 Nef downmodulates HLA-A and -B to protect infected cells from CD8(+) T cells but leaves HLA-C on the cell surface to inhibit NK cells. In this issue of Cell Host & Microbe, Apps et al. (2016) revise this model by showing that the Vpu protein of primary HIV-1 isolates downmodulate HLA-C., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure., Methods and Findings: Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I-presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control., Conclusions: These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.

Unlabelled: The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface., Importance: This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Tetherin serves as an innate sensor of viral infection in addition to its role in inhibiting virus release from infected cells. In this issue, Galão et al. (2014) provide important insights into the mechanism of virus-induced signal transduction by tetherin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1-specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor-mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.

Cellular proteins called "restriction factors" can serve as powerful blockades to HIV replication, but the virus possesses elaborate strategies to circumvent these barriers. First, we discuss general hallmarks of a restriction factor. Second, we review how the viral Vif protein protects the viral genome from lethal levels of cDNA deamination by promoting APOBEC3 protein degradation; how the viral Vpu, Env, and Nef proteins facilitate internalization and degradation of the virus-tethering protein BST-2/tetherin; and how the viral Vpx protein prevents the premature termination of reverse transcription by degrading the dNTPase SAMHD1. These HIV restriction and counter-restriction mechanisms suggest strategies for new therapeutic interventions.

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4(+) T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies-frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.

Tetherin (BST-2 or CD317) is an interferon-inducible cellular factor that prevents the detachment of enveloped viruses from infected cells. The primate lentiviruses have evolved different countermeasures to tetherin. The majority of SIVs use Nef to antagonize the tetherin proteins of their nonhuman primate hosts. However, due to the absence of sequences in human tetherin required for antagonism by Nef, HIV-1 Vpu and HIV-2 Env evolved to serve this function in humans. We recently identified compensatory changes in the Env cytoplasmic domain of a pathogenic nef-deleted SIV that confers resistance to rhesus macaque tetherin. These observations highlight the extraordinary plasticity of the primate lentiviruses in adapting to the tetherin proteins of their respective hosts, and reveal a prominent role for tetherin in shaping the evolution of the primate lentiviruses.

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.

Background: In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case-control analysis to identify antibody and cellular immune correlates of infection risk., Methods: In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up., Results: Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P=0.02; q=0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P=0.03; q=0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies., Conclusions: This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.

The interferon-inducible, transmembrane protein BST-2 (CD317, tetherin) directly holds fully formed enveloped virus particles to the cells that produce them, inhibiting their spread. BST-2 inhibits members of the retrovirus, filovirus, arenavirus and herpesvirus families. These viruses encode a variety of proteins to degrade BST-2 and/or direct it away from its site of action at the cell surface. Viral antagonism has subjected BST-2 to positive selection, leading to species-specific differences that presented a barrier to the transmission of simian immunodeficiency viruses (SIVs) to humans. This barrier was crossed by HIV-1 when its Vpu protein acquired activity as a BST-2 antagonist. Here, we review this new host-pathogen relationship and discuss its impact on the evolution of primate lentiviruses and the origins of the HIV pandemic., (Published by Elsevier Ltd.)

A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective -35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the "protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective -35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the -35TT genotype. Since the latter Nef functions all influence the stimulation of CD4(+) T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common -35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host-cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIV(smm/mac)/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIV(mac)239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIV(smm), HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.